Precise and accurate manipulation of nanolilter liquid and microliter liquid is very important to modern engineering science, physical science, chemical science, material science, pharmaceutical science, micromachining technology, and widely used in biochemical analysis, environment monitoring, medicine, clinical detection, drug screening, and nanomaterial synthesis and preparation technology. Generating independent water-in-oil or oil-in-water microdroplets is very important to the microliquid manipulation. Multitudinous micro-reactions and micro-screenings can be realized base on the microdroplets. Multitudinous and uniform microdroplets can be prepared by emulsion polymerization (referring to Bovey F A et al., Emulsion polymerization, New York: Interscience publishers, 1955, 1-22), membrane emulsification (referring to Nakahsima T et al., Membrane Emulsification by microporous glass, Key Engineering Mater, 1991, 513:61-61), and spay emulsification (referring to Liu. Y et al., Mixing in a multi-inlet vortex mixer (MIVM) for flash nano-precipition, Chemical Engineering Science, 2008, 63(11):2892-2842). The above methods are mainly applied in microsphere preparation and drug carrier preparation. However, volumes of the microdroplets cannot be precisely and accurately controlled. Therefore, these methods are not suitable for a microreactor or complicated biochemical reaction which require precise and accurate control of the volumes of the microdroplets.
Droplet generating method based on microfluidics (referring to The S Y et al., Droplet microfluidics, Lab on a Chip, 2008, 8(2):198-220) has been developed rapidly in recent years. A microdroplet can be generated in a microchannel of a microfluidic chip based on an unstable interface between a dispersion phase and a continuous phase when they meet in the microchannel. Through different designs of the microfluidic channel, uniform microdroplets can be generated, merged, reacted, and screened. However, the volumes of the microdroplets are limited by the structure of the microchannel and a surface feature modification of the microchannel. In addition, the microdroplets generated in the microchannels must be transferred to a storage container by specific device and method, which increases a difficulty to locate, extract, and analyze the microdroplets.
A simple method used to generate the microdroplet, comprises ejecting or spraying a liquid into a microwell or spotting a liquid on a substrate by a capillary micro-pipe or capillary, for short. However, when the microdroplet is released from the capillary, it is difficult to precisely control the amount of microdroplets due to a surface tension between the liquids inside and outside the capillary, and an adhesion force between the microdroplet and an orifice of the capillary. To overcome the surface tension, piezoelectric ceramics, thermal expansion, high voltage electronic injection, and ultrasound are used to increase a kinetic energy of the microdroplet. To decrease the adhesion force, the structure of an outlet end of the capillary is modified, and the surface of the capillary is coated or silanized. However, complicated and expensive liquid driving devices are used in these methods, and the volumes of the microdroplets are difficult to control directly or defined.
Analysis technology based on digital single molecules and single cells has been developed in recent years. Uniform microdroplets are ideal carriers of the single molecules and the single cells used in quantitative reaction and analysis. A digital nucleic acid molecule amplification technology (e.g. digital polymerase chain reaction, dPCR) is a representative digital nucleic acid quantitative analysis technology, wherein nucleic acid molecules in a sample solution are distributed to a plurality of micro-reaction systems according to the Poisson distribution, each micro-reaction system substantially comprises either one nucleic acid molecule or no nucleic acid molecule, independent amplified reaction is carried out in each micro-reaction system, and the nucleic acid molecules are quantified by counting a number of positive micro-reaction systems. The digital nucleic acid molecule amplification technology is especially suitable for studying variation in counts of gene sequence, such as copy number variation and point mutation.
Digital enzyme linked immunosorbent assay (Digital ELISA) technology which is similar to the digital nucleic acid quantitative analysis technology is also developed in recent years (referring to Rissin, D. M. et al., Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations, Nature Biotechnology, 2010, 28, 595-599), wherein microspheres containing immune antibodies are put into a femtoliter microwell array, single target protein molecules are selectively combined with the immune antibodies, the microwell array is imaged by enzyme-linked immunoassay, and the single target protein molecules are quantified by counting a number of microwells whose fluorescence signal is amplified.
Digital single cell analysis is one of the hottest fields in modern biology. The digital single cell analysis is an important method to detect cell heterogeneity which is difficult to be found by conventional method, analyze compositions of cell subset, find low-probability cell mutation, and explore uncultured microorganisms. The single cell is too tiny to be manipulated. The single cell of bacteria, fungus, plants and animals can be enveloped in the microdroplet with a diameter ranging from several tens of microns to several hundreds of microns. The microdroplet can supply a micro-environment for the single cell which is convenient to be manipulated. The extracellular materials secreted by the single cell can be gathered quickly in picoliter or nanoliter microdroplets. In addition, multitudinous single cell cultures, enzymatic activity analysis of the single cell, single cell analysis, genome amplification of the single cell, and transcriptome amplification of the single cell can be realized in the microdroplet.
After a molecule solution or a cell solution is mixed with reactants, the mixture can be distributed into a plurality of microsystems. A single molecule or a single cell can be reacted, grown, and amplified in each microsystem, after which an array detection and a digital analysis can be applied to the plurality of microsystems, which improves reliability and sensitivity of biological detection and clinical diagnosis, and has a wide application prospect.